This project focuses on the creation of a model flavoprotein for the detailed investigation of the determinants of redox potential tuning in flavoenzymes, a family of redox proteins implicated in many genetic diseases. A bilateral approach using both synthetic iterative redesign and phage display directed evolution methodologies will be used to generate a highly stable, high-affinity model flavoprotein. This model protein will allow the unique opportunity of examining in depth the dynamic details of proton and electron transfers and their coupling in flavoproteins. The thermodynamics of the bound flavin will be investigated potentiometrically. Electron transfer rates and rate-determining proton transfer rates will be measured as a function of pH using fast scan protein film voltametry. Amino acid residues involved in the delivery of protons to the flavin during reduction will be identified using solution NMR. A quantitative global analysis of the extraordinarily large body of flavoprotein structures and reduction potentials which have been published will be performed to provide a set of engineering principles which will aid in the construction of flavoprotein maquettes with a range of redox properties. These principles will be demonstrated by the incorporation of specific flavin-protein interactions into the model flavoprotein followed by the determination of the changes induced in the bound flavin?s redox properties. As proton transfer events are an intrinsic part of flavin redox chemistry, understanding the coupling between electron and proton transfer in these model proteins is a crucial step towards a greater comprehension of redox potential tuning in flavoproteins.